2c7q: Difference between revisions
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==Overview== | ==Overview== | ||
DNA base flipping is an important mechanism in molecular enzymology, but | DNA base flipping is an important mechanism in molecular enzymology, but its study is limited by the lack of an accessible and reliable diagnostic technique. A series of crystalline complexes of a DNA methyltransferase, M.HhaI, and its cognate DNA, in which a fluorescent nucleobase analogue, 2-aminopurine (AP), occupies defined positions with respect the target flipped base, have been prepared and their structures determined at higher than 2 A resolution. From time-resolved fluorescence measurements of these single crystals, we have established that the fluorescence decay function of AP shows a pronounced, characteristic response to base flipping: the loss of the very short (approximately 100 ps) decay component and the large increase in the amplitude of the long (approximately 10 ns) component. When AP is positioned at sites other than the target site, this response is not seen. Most significantly, we have shown that the same clear response is apparent when M.HhaI complexes with DNA in solution, giving an unambiguous signal of base flipping. Analysis of the AP fluorescence decay function reveals conformational heterogeneity in the DNA-enzyme complexes that cannot be discerned from the present X-ray structures. | ||
==About this Structure== | ==About this Structure== | ||
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[[Category: transferase]] | [[Category: transferase]] | ||
''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 16:45:52 2008'' | ||
Revision as of 17:45, 21 February 2008
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HHAI DNA METHYLTRANSFERASE COMPLEX WITH OLIGONUCLEOTIDE CONTAINING 2-AMINOPURINE OUTSIDE THE RECOGNITION SEQUENCE (PAIRED WITH G) AND SAH
OverviewOverview
DNA base flipping is an important mechanism in molecular enzymology, but its study is limited by the lack of an accessible and reliable diagnostic technique. A series of crystalline complexes of a DNA methyltransferase, M.HhaI, and its cognate DNA, in which a fluorescent nucleobase analogue, 2-aminopurine (AP), occupies defined positions with respect the target flipped base, have been prepared and their structures determined at higher than 2 A resolution. From time-resolved fluorescence measurements of these single crystals, we have established that the fluorescence decay function of AP shows a pronounced, characteristic response to base flipping: the loss of the very short (approximately 100 ps) decay component and the large increase in the amplitude of the long (approximately 10 ns) component. When AP is positioned at sites other than the target site, this response is not seen. Most significantly, we have shown that the same clear response is apparent when M.HhaI complexes with DNA in solution, giving an unambiguous signal of base flipping. Analysis of the AP fluorescence decay function reveals conformational heterogeneity in the DNA-enzyme complexes that cannot be discerned from the present X-ray structures.
About this StructureAbout this Structure
2C7Q is a Protein complex structure of sequences from Haemophilus haemolyticus with , and as ligands. Active as DNA (cytosine-5-)-methyltransferase, with EC number 2.1.1.37 Known structural/functional Site: . Full crystallographic information is available from OCA.
ReferenceReference
Time-resolved fluorescence of 2-aminopurine as a probe of base flipping in M.HhaI-DNA complexes., Neely RK, Daujotyte D, Grazulis S, Magennis SW, Dryden DT, Klimasauskas S, Jones AC, Nucleic Acids Res. 2005 Dec 9;33(22):6953-60. Print 2005. PMID:16340006
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